1. Field of the Invention
The present invention relates to a color-forming coupler which can be used in test compositions, methods and apparatus for detection of hydrogen peroxide. In the presence of peroxidase and hydrogen peroxide, the couplers of the present invention react with a hydrazone or an aminoantipyrine to produce a deeply colored dye, which can be used to give a visual indication of the analyte present in the sample. These couplers can be used in diagnostic tests in which the signal producing system generates hydrogen peroxide as a measure of the analyte present.
2. Description of the Background Art
The detection and quantitative determination of hydrogen peroxide, and compounds capable of reacting to form hydrogen peroxide, are important in many areas. For example, hydrogen peroxide is produced in the enzymatic assay of various substances, such as glucose, cholesterol, uric acid, and the like, through the activity of enzymes such as glucose oxidase, cholesterol oxidase, uricase, and the like. The quantity of enzyme substrate present in a sample is determinable from the amount of hydrogen peroxide produced and detected.
Compositions for detecting and/or quantifying hydrogen peroxide in such systems generally comprise a substance having peroxidative activity, such as peroxidase and peroxidase-like substances, and material which undergoes a detectable change, generally a color change, in the presence of hydrogen peroxide and the peroxidative substance. There are many compositions which can be used for such determinations, including mono- and diamines, phenols, polyphenols, aromatic acids, dyes, and other compounds which produce colors under the conditions sought to be detected.
There are many examples of color-forming substrates of peroxidase and peroxidase-like substances which have been previously suggested, including the following substances such as monoamines, diamines, phenols and polyphenols, aromatic acids, dyes including leuco dyes, flayones, epinephrine, gum guaiac, guaiconic acid, water soluble iodides, and bilirubin.
Although the above-named substances are generally useful as indicator systems for the detection of hydrogen peroxide, there are instances in which the concentration of hydrogen peroxide to be analyzed is too low to produce sufficient detectable color from these indicators, e.g., because of the source of the hydrogen peroxide, the necessity for dilution, or the overall detection method. A specific example of such a concentration problem occurs in the detection of hydrogen peroxide produced from the low levels of uric acid present in blood serum (on the order of 1-15 mg/dl). This problem is conventionally ameliorated by measuring relatively large volumes of the detectable product, e.g., by increasing the diameter of the cuvette in a solution assay for uric acid so as to additively increase the relative density of the color produced. However, this solution can only be used when the assay is conducted in a cuvette.
All analytical techniques do not permit the use of such modifications to increase the effective density of the indicator produced. This is a particular problem for use in dry chemistry assay devices, where only a limited amount of color-forming substrate can be incorporated in a strip-type device. In other assays, such as, in solution, the necessity for high dilution levels to obtain proper dissolution or the like, may make it impractical to use increased concentrations of dye.
These problems are particularly acute when the analyte determination is performed in a dry strip assay device such as a multilayer element in which the reagents are impregnated in or coated onto the layers which receive the sample. In these cases, using the relatively thin indicator or reagent layers (on the order of less than one mil) that are desirable in these devices, the density of the color formed can be rather low. Increasing the thickness of the color- forming layer to provide greater density many be undesirable, as it can increase reaction times, create problems in layer preparation, etc. Consequently, other techniques for increasing the effective density of the dye produced in the indicator are necessary, if this type of device is to be used reliably for the assay of low concentration blood serum components. Moreover, in quantitative assays, it may be undesirable to increase the amount of color former beyond a certain level, as this may interfere with the quantitative detection of the analyte.
A number of workers in this field have sought to overcome the problems associated with detection of hydrogen peroxide, and have met with varying degrees of success. Bruschi in U.S. Pat. No. 4,089,747 and Lam, in U.S. Pat. No. 4,119,405, relate to assays for hydrogen peroxide or analytes which generate hydrogen peroxide using a combination of a hydrazone and a color coupler. Use of the hydrazones described in these patents in assaying whole blood has the disadvantage that it is difficult to obtain detectable dyes which absorb electromagnetic radiation at relatively long wavelengths, i.e., greater than about 600 nm. Dyes formed with hydrazones generally absorb at shorter wavelengths and their detection is often hindered by various spectral interferents which are present in whole blood sample. The presence of these interferents, of course, diminishes the accuracy of the assay using dyes formed from hydrazones in testing whole blood.
Evans et al., in U.S. Pat. No. 4,737,457, use a hydrazide, or a hydrazine substituted with at least one electron withdrawing group; these compounds react with phenolic color couplers in the presence of hydrogen peroxide and a peroxidative compound to form a dye which is detectable at a wavelength of 600 nm or greater.
Lam, in U.S. Pat. Nos. 4,071,317; 4,071,318; and 4,071,321, discloses test compositions and devices for detecting peroxidatively active substances. These compositions are based upon the use of borate esters, and are used for dip stick devices which provide a qualitative detection of a pseudo peroxidative substance such as hemoglobin to detect occult blood.
Mast et al., in U.S. Pat. No. 3,986,833, disclose test compositions for detecting peroxidatively active substance including an indicator capable of being oxidized in the presence of a peroxidatively active substance to provide a color change, an oxidizing agent effective to oxidize the indicator, and a potentiating agent. The potentiating agent is a water soluble acid salt or adduct of a quinoline which may be substituted by lower alkyl, hydroxy, thiophenyl, carboxy or lower alkoxy groups.
Washburn, in U.S. Pat. No. 4,672,029, discloses color-forming couplers for combination with an oxidizable color developing compound, such as 4-aminoantipyrine, to provide a chromogenic composition.
Klose et al., in U.S. Pat. No. 4,101,381, describe a dye for photometric detection of hydrogen peroxide. In this case, the dye is formed using as the hydrazone 3-methyl-2-sulfonylbenzothiazolone-hydrazone. The dye formed by this compound is said to be easily soluble and suitable for use in automatic analyzers, and forms a dye which is measured at 620 or 670 nm. The assay reactions take place in solution, so that the system is not suitable for use in making dry chemistry strips for quick and reliable assays.
Schmitt et al., U.S. Pat. No. 3,770,381, describe dipsticks for semiquantitative colorimetric detection of organic peroxides. The method described therein requires immersing the dipstick in a solution of peroxides in a volatile organic solvent having a boiling point of below 80.degree. C. and withdrawing the dipstick. The dipstick is then placed in a water vapor atmosphere having a relatively constant temperature between about 20.degree. and 100.degree. C. to evaporate the organic solvent and deposit moisture on the surface to produce a color change. This procedure, of course, requires the use of solvent vapor as well as water vapor, and is certainly not convenient to use.
Katsuyama et al., in U.S. Pat. No. 4,418,037, use pyrogallol derivatives to stabilize film used for detecting hydrogen peroxide colorimetrically. The dyes, which must be stabilized, are formed from a hydrogen donor and a coupler.
Yamanisi et al., in U.S. Pat. No. 4,251,629, disclose a composition for colorimetric determination of hydrogen peroxide comprising 4-aminoantipyrine, a N-substituted-3-alkylaniline and a hydrogen peroxide activating agent such as peroxidase. The condensation of the 4-aminoantipyrine with the N-substituted-3-alkylaniline in the presence of hydrogen peroxide produces an indophenol type dye which has a maximum absorption near 545 nm. These dyes are used in solution assays rather than dry chemistry strips.
Babb, in U.S. Pat. No. 4,670,385, discloses triarylmethane leuco dyes which have an aromatic heterocyclic moiety attached to the central methane carbon and which, upon interaction with hydrogen peroxide, provide dyes which absorb at wavelength at least about 600 nm. Because the leuco dyes have limited solubility in water, test compositions or devices using these dyes must be prepared with solvents such as alcohol or N,N- dimethylformamide.
Nix et al., in U.S. Pat. No. 4,247,631, disclose the use of a colored dye based upon the reaction product of a 3- aminobenzoic acid or a 3-aminobenzenesulfonic acid with a 4-aminoantipyrine in the presence of hydrogen peroxide. The dyes formed by this reaction generally have absorption maxima in the range of from about 450-650 nm.
Berti et al., U.S. Pat. No. 4,247,297, disclose indicator dyes useful to test devices comprising a hydrazone and 8-amino-1-naphthol-5,7-diosulfonic acid. Hydrogen peroxide can be quantitatively detected with these compositions using a conventional spectrophotometer at wavelengths ranging from about 490 nm to about 620 run. As with a number of the other assay systems described above, these dyes are not readily usable in a dry reagent strip format.
Bouse et al., U.S Pat. No. 4,971,918, disclose indicator compositions containing a pyrogallol derivative along with a reducible or charge carrier complex forming chromogen. In this case, the indicator system does not require a coupling compound.
Trager et al., in U.S. Pat. No. 4,492,754, disclose a composition for the detection of hydrogen peroxide or hydrogen peroxide forming substrates using a substrate capable of coupling is a compound of the formula: ##STR1##
wherein n and m are individually selected from whole numbers 1 to 4,
X and Y, which can be the same or different, represent a valence bond or a phenylene radical,
R.sub.1 and R.sub.2 are individually selected from carboxyl or sulfonic acid groups and one of R.sub.1 and R.sub.2 can also be hydrogen or lower alkyl, and
R.sub.3 and R.sub.4 are individually selected from hydrogen and alkyl radicals of up to 6 carbon atoms.
These aniline derivatives contain both an acid and a basic group, and are normally present as internal salts or as alkali metal or ammonium salts. Colors formed with these compounds must be read photometrically.
Tsuda et al., in U.S. Pat. No. 4,260,679, disclose reagents for quantitative determination of hydrogen peroxide comprising a hydrogen donor of the formula: ##STR2##
Where R.sub.1 is CH.sub.3, C.sub.2 H.sub.5, CH.sub.2 OH, CH.sub.2 CH.sub.2 OH, CH.sub.2 CH.sub.2 NH.sub.` or COCH.sub.3 ;
R.sub.2 is CH.sub.2 OH, CH.sub.2 CH.sub.2 OH, CH.sub.2 CH.sub.2 NH.sub.2, CH.sub.2 NHCOCH.sub.3, CH.sub.2 CH.sub.2 NHCOCH.sub.3, or COCH.sub.3 ;
R.sub.3 is H, CH.sub.3, C.sub.2 H.sub.5 or OCH.sub.3 ; and
R.sub.4 is H or CH.sub.3.
Electron acceptors used with these compounds are conventional materials such as 4-aminoantipyrine, 2-thio- phenecarboxylic acid hydrazide, benzidine or 3-methyl -2- benzthiazolinione hydrazone.
No admission is made that any reference cited herein constitutes prior art, and applicants reserve the right to challenge the nominal publication date or alleged teachings of any cited reference.